A Ca2+ channel differentially regulates Clathrin-mediated and activity-dependent bulk endocytosis (PLOS Biology, 2017)

It has been known that a tight coupling ofexocytosis and endocytosis plays a crucial role in sustaining synaptic transmission. Moreover, Clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE) are the
two predominant forms of synaptic vesicle endocytosis, triggered by moderate and strong nerve stimuli, respectively. Multiple lines of evidence suggest that exocytosis and intracellular Ca2+ elevation are prerequisites for both modes of endocytosis to proceed. Nevertheless, it remains unclear how the two events cooperate to promote endocytosis upon exocytosis. Our findings indicate that the synaptic vesicle-associated Ca2+ channel Flower we previously identified has a
significant role in these coupling processes. We found that Flower initiates ADBE in addition to CME. Most importantly, the effects of Flower depend on the strength of stimulus. Thus, we point out a basis for the differences between CME and ADBE. Our results reveal that Flower physically and genetically interacts with Dynamin and promotes its localization at Clathrin-coated pits, a process required for SV fission in CME. Surprisingly, this function does not involve Ca2+ channeling by Flower. Strikingly, upon intense stimulation, Flower triggers a Ca2+ influx that elicits ADBE. This finding therefore identifies the Ca2+ source for ADBE, which was previously unknown. We further assessed the role of Flower across species by knocking out rat Flower homolog with CRISPR/Cas9 technology in cultured rat hippocampal neurons. We show that the endocytic role of Flower is conserved at mammalian central synapses. Overall, Flower plays a universal role in promoting CME and ADBE. We therefore propose that the Flower channel can exert two different functions in response to two different stimuli to govern distinct modes of synaptic vesicle retrieval, thereby coupling exocytosis to endocytosis. Our findings not only reinforce the importance of Ca2+ in synaptic vesicle retrieval but also address how different modesof endocytosis respond to different nerve stimuli and are coupled with exocytosis.